Antibody Catalyzed Water Oxidation Pathway Biosensor

Infectious diseases, such as influenza, present a significant global challenge through the constant threat of pandemics. We report the development of a novel biosensor that can be functionalized to address a wide range of infectious diseases. This biosensor is based on the electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the Antibody Catalyzed Water Oxidation Pathway (ACWOP). Our biosensor platform includes a polymer brush-modified surface where antibodies bind to a conjugated small molecule, or hapten, that is capable of eliciting an immune response. The antibody produced hydrogen peroxide supplies the electrochemical signal mediated by Resorufin/Amplex Red. We demonstrate the complete function of the biosensor platform using anti-2,4-dinitrophenyl (anti-DNP) antibodies as a model system. Currently, the standard method for antibody detection is based on the enzyme-linked immunosorbent assay (ELISA) which requires labeled secondary reagents to bind antibodies, as well as multiple procedural steps. Thus, the ultimate goal of the biosensor described here will involve preparing an inexpensive device that is portable, reliable, and fast.

Antibody Catalyzed Water Oxidation Pathway Biosensor

Infectious diseases, such as influenza, present a significant global challenge through the constant threat of pandemics. We report the development of a novel biosensor that can be functionalized to address a wide range of infectious diseases. This biosensor is based on the electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the Antibody Catalyzed Water Oxidation Pathway (ACWOP). Our biosensor platform includes a polymer brush-modified surface where antibodies bind to a conjugated small molecule, or hapten, that is capable of eliciting an immune response. The antibody produced hydrogen peroxide supplies the electrochemical signal mediated by Resorufin/Amplex Red. We demonstrate the complete function of the biosensor platform using anti-2,4-dinitrophenyl (anti-DNP) antibodies as a model system. Currently, the standard method for antibody detection is based on the enzyme-linked immunosorbent assay (ELISA) which requires labeled secondary reagents to bind antibodies, as well as multiple procedural steps. Thus, the ultimate goal of the biosensor described here will involve preparing an inexpensive device that is portable, reliable, and fast.

Judges’ Queries and Presenter’s Replies

While we have not observed a false positive or negative, likely sources could be an uncharacteristically high concentration of hydrogen peroxide or a radical inhibitor already present in the sample. Depending on the sample, it may be possible that other biological material may interfere with our detection of hydrogen peroxide, but we have found nothing to suggest that that might happen.

A limitation with our technique may include the fact that extended exposure to UV light is needed for optimal singlet oxygen production. We have currently tested irradiation for 60 minutes and have found that longer times can result in a signal decrease and an increase in background, possibly due to UV damage of bound antibodies. However, we have been using a wide benchtop UV light source. It may be likely that a more concentrated light source could serve to shorten this temporal requirement while minimizing any antibody damage.

Other weaknesses are currently in the necessity of intermediates and additional reagents, like Amplex Red, for the detection of antibody produced hydrogen peroxide. For a field ready and portable biosensor device, it may be important to consider alternative detection schemes. Yet, the Amplex Red assay has been widely tested as a highly specific and sensitive measure of hydrogen peroxide concentrations. Also, the Resorufin product from this assay is very stable, allowing quantification of hydrogen peroxide in both oxidative and reductive conditions. Combining the Amplex Red assay with square wave voltammetry in our biosesnor, low hydrogen peroxide concentrations are currently detectable with high fidelity.

Does your biosensor need to be adapted or configured to work properly in different environments? For example, does the very high temperature in a remote village in Africa prevent the application of your biosensor in field disease studies? Does the small age of the patient hamper detecting infection in newly born infants?

Higher temperatures would promote a faster production of hydrogen peroxide while allowing for a larger signal from bound antibodies. Conversely, colder temperatures would generate a lower signal but still allow for sensitive detection. It is possible to modify the polymer brushes with other small molecules for a wider range of antibody binding. In addition, the samples would all be at a temperature of approximately 37 degrees Celcius regardless of different environments.

Small age should not be a problem for our biosensor as it uses only a minimal sample volume to be able to detect picogram quantities of the antibody.

Thank you Dr. Cowles for your question.
Our biosensor compares quite favorably with standard ELISA techniques. Current tests demonstrate that hydrogen peroxide at concentrations as low as 0.33 nM can be measured. Antibodies bound at ~ 5 × 10^-12 mol/cm^2 produce over 25 × 10^-10 mol H2O2/cm^2, which translates to above 250 mM of hydrogen peroxide generated from only 10 µL of a test sample. Stated another way, these results indicate that below 3 picograms of bound antibody may be detected from a 10 µL sample, which equates to an antibody concentration of just 2 pM.
Further, standard ELISA techniques rely on large solid supports (typically a microtiter plate) for immobilizing hapten molecules with either non-specific adsorption or by specifically capturing hapten via surface coated antibodies. Our biosensor, on the other hand, avoids challenges with these approaches by using a smaller surface area by comparison, for the presentation of sufficiently high densities of covalently attached hapten (e.g. DNP) to polymer brushes. Enhanced specificity with our biosensor comes through the capability of modifying these brushes to present a wide range of antibody haptens. Additionally, the polymer brush OEG moieties resist non-specific binding and have long-term stability as a result of their high packing characteristics. We have incubated non-specific antibodies on our DNP capped brushes and photosensitizer surfaces and have found no significant change in frequency as detected by QCM measurements, confirming that non-specific antibodies do not bind to the polymer brushes.

Really great work guys! Your video is so well depicted and explained! This seems like a really awesome system, but I’m wondering if you need to use a special method in order to get the polymer brushes to extend up from the surface with their functional groups exposed to the antibody rather than laying flat in a jumble.
Thanks for the insightful and fun explanations!

Thanks Angie! To answer your question, the OEG moieties of the polymer brushes have high grafting densities, making them resistant to non-specific binding while offering long-term stability. More specifically, it is this dense packing of neighboring polymer chains that results in an increased entropic force to drive the brushes into a stretched, upright state.

Thank you Myisha for your question.
The development of our biosensor has been focused primarily on the detection of antibodies specific to pathogens like influenza. However, the detection of bacteria and fungi should also be feasible. Provided that our polymer brush surface can be functionalized to present the appropriate haptens (small molecules) to bind bacterial and fungal antibodies, we should be able to identify the presence of these organisms from a test sample.

David Bradford

Guest

May 22, 2013 | 08:00 p.m.

Nice job guys. Your video was remarkably understandable. It was presented so well that even an old guy like me, who hasn’t studied high level molecular biology in such a long time, grasped the idea of what your were doing. Have you developed a prototype that will allow you to take your work out into the field to test it? To think I knew you Devin when you didn’t know anything about chemistry. Way to surpass your teacher!
P.S. I think one of your commenters is a little biased.

Thanks Dr. Bradford!! I am so glad you enjoyed our video.
Our biosensor at present consists only of the patterned quartz crystal microbalance (QCM) or patterned silicon chips. The QCM has allowed us to thoroughly characterize the photosensitizer / polymer brushes, determine the extent of antibody binding, and measure our limits of hydrogen peroxide detection. Having confirmed the successful performance of these aspects in the lab, we are now looking into the development of a device to enable field testing. We are considering a microfluidic platform capable of handling small fluid and reagent volumes. This system could be tuned to provide the right balance of photosensitizer and polymer brushes (covering the inside of microfluidic channels), while electrochemical features (e.g. microelectrodes) could be selectively incorporated to supply optimal hydrogen peroxide detection.
P.S. I agree, one of my commenters has been eagerly awaiting the chance to view and comment on my work :)

Serena Ambrosini

Guest

May 23, 2013 | 01:33 a.m.

Dear Devin congratulation for your work!
The universabilty of this system is quite attractive!
Since I am working on molecularly imprinted polymers (MIPs), what do you think about using MIP-based sensing layer?
This would not be applicable to all kinds of pathogens..but it will offer an higher specificty and stability.

Thanks Serena!!
I think the applications of MIPs for our biosensor would be quite interesting. Provided good compatibility with the patterning approach and photosensitizer, MIPs with the necessary functionality for antibody binding could be readily prepared. I would be interested to learn how the specificity and stability of MIPs compare to that of our current polymer brushes.